LZU-HS-Pro-A

General SAFETY

Covid-19 problem

Here is LZU-HS-Pro-A team, the team members have signed the laboratory safety guarantee regulations before entering the laboratory, and presented a 24-hour nucleic acid certificate. Also, since there are much more professional experimental labs in Beijing than other places and most of our team members could successfully get to there and don’t need to be isolated. Even though there are some members could get into the lab, they still can do the experiment online in other province or countries. Di Guan, who is the manager of security at the lab, and our school has a laboratory safety management department.

Nucleic acid amplification testing

Lab Online

Safety on experiment

We attach great importance to the safety guarantee during the team experiment. We have entered the Beijing Center for Physical & Chemical Analysis, which has advanced equipment and strict codes of conduct. Entering the laboratory, a doctoral tutor led us to do experiments. The probiotic used by the team was Nissle 1917. This type of Escherichia coli is the only non-pathogenic Escherichia coli, and we try to use as little dangerous experimental equipment as possible during the experiment. This will greatly avoid experimental errors.

Beijing Center for Physical and Chemical Analysis

Laboratory Safety Information Sheet

Laboratory Safety Inspection Checklist

When we use nucleic acid electrophoresis, the electrophoresis contains EB, which is a strong mutagen that can cause cancer or teratogenicity. Therefore, we will wear standard lab coat and two layers of gloves, one layer of medical examination gloves and one layer of PET gloves, because PET gloves are resistant to fat, dilute acid, dilute alkali, and most solvents, so this can well protect our body from direct contact Touch something in electrophoresis.

EtBr (EB)

Latex gloves

standard lab coat

And we also learned the professional way of removing gloves which improved the level of safety even better. In order to prevent the experimental liquid from being splashed on the body, the team members will wear professional special protective experimental clothes to enter the laboratory. In order to protect the eyes from the invasion of experimental foreign bodies, we will wear professional medical goggles. Try not to touch the items in the nucleic acid pool during the electrophoresis practice. Due to the toxicity of EB, after the experiment, the solution containing EB should be purified and disposed of to avoid polluting the environment and endangering human health.

Proper steps for removing gloves in the lab

Professional medical goggles

The team is also very careful when using the centrifuge. We will always pay attention to the speed and temperature of the centrifuge. When putting reagents for centrifugation, we will be very careful to balance them, and pay attention to the direction of each E tube. When we use the biological safety cabinet, we will ensure a sterile environment in the cabinet. After using the alcohol lamp, even if it is pressed off, close the glass cabinet door after the experiment is completed. After the experiment, we will discard the generated experimental waste into the biological trash can.

the centrifuge

biological safety cabinet

Precautions for the use of biological safety cabinets and removal of contaminati from biological safety cabinets

Project safety

Avoiding GMO contamination

We decided to use more common small molecule operation. son. Under normal conditions, the arabinose promoter initiates the expression of the latter three enzymes when arabinose occurs to decompose arabinose. Our plan is to insert an anti-toxin gene after the production of these three enzymes, and then express a toxin gene after. Arabinose is present in the small intestine and is not broken down by the small intestine. Therefore, when E. coli is in the small intestine, it can receive the arabinose in the small intestine, express the later anti-toxin gene, and neutralize the later expressed toxin gene, and the bacteria can survive normally. When E. coli leaves the small intestine, the arabinose operon without arabinose regulation does not express the following antitoxin genes, only the toxin genes are expressed. Bacteria complete suicide. After consulting related papers, the toxin gene kid and anti-toxin gene kis contained in the toxin-antitoxin system parD of plasmid R1 were selected as the main components of the suicide system. The specific assumption is as shown in the figure below. In the original arabinose manipulation On the basis, in the last D region expressing the epimerase, insert a promoter specifically expressing the kis antitoxin gene [3], and insert the kis gene behind to express the P10 antitoxin protein kis[4] (in the presence of arabinose). case) insert the promoters that start the kid and kis genes and the kid gene of the toxin protein after the kid gene. After the design was completed, we further modified nissle 1917 and completed functional testing of the gene suicide system.

After reviewing more papers, we finally found a relatively complete two CRISPR-based Nissel 1917 kill switches in Structure and function of bacterial kid-kis and related toxin-antitoxin systems, one as a chemical response switch and input chemical and temperature responsive switches, while improvements have been made for switch stability. A chemically responsive switch initiates a death switch in response to the chemical inducer anhydrotetracycline (aTc), and a kill switch initiates additional death in response to a temperature dip caused by host excretion For the chemical switch, it is based on the design of CRIPER, cas 9 and gRNA are expressed through the aTc-inducible promoter Ptet, and TetR needs the promoter Ptet to be expressed after the gRNA. In the presence of aTc, TetR cannot bind to the target promoter, and the cell will initiate the Pet promoter to express cas9 and gRNA. Select the optimized 2-gRNA for expression (it can effectively reduce manslaughter and improve stability).

Four genes, recA, polB, dinB and umuDC, need to be knocked out if the SOS response induced by single DNA DSB induced by CRIPER-Cas 9 occurs. At the same time, in the in vitro simulated small intestine environment and animal model experiments in this article, it was found that the ecology in the intestine can often prevent the re-reproduction of escape mutants, and can maximize the killing range.

Suicide system engineering figure

Safe entry of strains into the human body

Coating technology of Escherichia coli:

Since the advent of the probiotic bacteria coating technology, it has been widely used in various microecological probiotic preparations. However, most probiotic preparations are embedded in three layers. In the actual application process, due to the high pH value of the human digestive tract, the strength of the wall material embedded with probiotics is low. A large number of bacterial flora appeared to die. In order to solve this problem, after many experiments, the researchers finally found that the use of probiotic strain self-embedding technology can maximize the protection of the probiotic activity from being destroyed, so that it can resist gastric acid and choline smoothly enter the intestinal tract. After entering the intestinal tract, probiotics are released in a specific environment in the intestinal tract, so that a large number of live bacteria can successfully colonize the intestinal tract to play a role. The unique multi-layer probiotic encapsulation technology can be used for the strain to protect the probiotics, so that the probiotics can play a greater role in human health. Multilayer embedding technology includes probiotic membrane, lipid membrane and polymer compound membrane to protect E. coli, which can be resistant to high temperature, saliva, bile and other digestive fluids. Based on this theory, we have designed a special coating.

multi-layer probiotic encapsulation

Safety in HP

Policies of different countries to protect local people from GMO contamination

Established the Regulations on the Safety Management of Agricultural Genetically Modified Organisms, implemented the safety management of agricultural biological genetic engineering, and established the "Regulations on the Safety of Genetically Modified Foods": comprehensive regulations on the safety of genetically modified foods, requiring that all foods from or containing genetically modified ingredients must be labeled. China conducts safety evaluation according to five stages: experimental research, intermediate test, environmental release, production test and application for safety certificate, and it will be terminated immediately after any unsafe problem to health and environment is found at any stage.

GMO-related laws in China

General process of genetically modified crops management

modified crops management

However, the existing legislative level is not high, the main body of responsibility structure is not obvious, and the food standard setting is low and conflicting with each other. At the same time, its labeling system is not perfect, and the lack of tracking system is too light to punish food safety offenders.

The main content and core are the Federal Food, Drug and Cosmetic Act, the Poison Control Act and the Plant Protection Act

The testing standards are the U.S. Food and Drug Administration (FDA), U.S. Environmental Protection Agency (EPA), and U.S. Department of Agriculture (USDA) ensure that GMOs are safe for human, plant, and animal health.

FDA regulates most human and animal food, including GMO foods. In doing so, FDA makes sure that foods that are GMOs or have GMO ingredients meet the same strict safety standards as all other foods. FDA sets and enforces food safety standards that those who produce , process, store, ship, or sell food must follow, no matter how the foods are created.

FDA regulation

EPA is responsible for protecting human health and the environment, which includes regulating pesticides. EPA regulates the safety of the substances that protect GMO plants, referred to as plant-incorporated protectants (PIPs), that are in some GMO plants to make them resistant to insects and disease. EPA also monitors all other types of pesticides that are used on crops, including on GMO and non-GMO crops.

EPA regulation

The USDA Animal and Plant Health Inspection Service (APHIS) protects agriculture in the United States against pests and disease. APHIS sets regulations to make sure GMO plants are not harmful to other plants, and USDA’s Biotechnology Regulatory Services implements these regulations.

USDA regulation

France On April 15, 2007, the French National Assembly voted to pass a bill submitted by the current government to ban the cultivation of Monsanto 810 genetically modified maize (MON810). In 2015, the planting of genetically modified crops and domestic production of genetically modified food were prohibited by legislation, and some genetically modified food was imported. Prohibition of planting MON 810 (In 2011, the European Court of Justice and the French Administrative Court ruled that the French Ministry of Agriculture's ban on MON 810 was illegal because it "failed to demonstrate a particularly high health and environmental risk”.) Products as long as the detection of genetically modified components, and the composition of more than a certain value must be genetically modified labeling. On September 17, 2015, the French government announced that it would effectively continue to ban genetically modified crops by enacting an "opt-out" clause.

French regulations on genetically modified products

Japan allows the production and sale of genetically modified food, but sets extremely high standards and strict treatment on GM cultivation. Following its accession to the Cartagena Protocol on Biosafety in 2003, Japan enacted the Cartagena Act of Japan, which deals with the environmental safety management of genetically modified organisms. The second is management regulations. For example, according to the Cartagena Law of Japan, corresponding management regulations are formulated according to the utilization mode and use of genetically modified organisms. Japanese model for GM food is one of "vertical regulation, supplemented by local regulation”. ach of the three organizations has vertical management of genetic modification with respect to their respective responsibilities. The food market has been implemented to distinguish the production and circulation management of genetically modified agricultural products as the main raw materials of food, and should be marked as "genetically modified food" The tendency toward production process-based management in Japan.

Inventory of Safety Evaluation of Genetically Modified Products in Japan

Canada has no special legislation for the safety of genetically modified organisms, so the regulation of the safety management of genetically modified organisms is scattered in the existing legal system. Canada's GMF-related laws include the Food and Drug Act, a series of laws to ensure people's safety. Adopt a voluntary labeling system, the government does not make mandatory provisions, the production and operators voluntarily choose whether to label the GM ingredients in their products (except for allergenic ingredients).Health Canada does not conduct any of its own testings on GM foods but relies on industry-generated data, except where there is any relevant science that also happens to be in the scientific literature.

Environmental Law in Canada

All GM foods sold in Australia and New Zealand must pass the safety evaluation of Food Australia New Zealand Authority, or they will not be allowed to be marketed. Australia has established a nationally uniform regulatory system for gene technology called the Gene Technology Act 2000, which came into effect on 21 June 2001 and covers any modification of genes or other genetic material (excluding sexual reproduction and homologous recombination). Provide that whether or not the genetically modified component is detectable in the final product should be identified. Many scientists have different answers to the safety issue, the state does not prevent it, but it must be checked, and there must be a GMO logo before it is sold. The company applying for the import or sale of GM food must submit an application to the relevant authorities and submit a list of GMOs used in the production of GM food, the characteristics of the food, the production method, etc. food characteristics, production methods, and a series of Detailed data and information. Innovation is the first driver of development and the principle of innovation

Safety evaluation of Food Australia New Zealand Authority

Safety Education

Not only to ensure the safety of the team members' experiments, we also hope that more people will pay attention to this, so we have opened online and offline education courses. For online activities, we have designed a lot of activities, such as moving pictures, cartoon videos, interesting copywriting, etc., and also carried out the promotion of experimental safety rules for common experiments, so that more people are aware of the importance of experimental safety.

Online video animation lectures

Online official account courses

The offline activity is located in the International Department of Beijing No. 2 Middle School. The wet leader of our team spreads this kind of safety knowledge to the students in the school. In the end, with the online and offline efforts of the team, we have a good understanding of the vast number of netizens and offline schools. The students in the school gave an effective and meaningful experiment safety lecture, and the participants benefited a lot. They learned about the safety measures of our specific projects, the protection and measures for depression, and finally the behavioral norms and precautions for general experiments.

Offline laboratory safety theory teaching

Offline using of laboratory equipment teaching

Intestinal alliance

We formed a gut consortium with other IGEM teams，HZ-CHINA, LZU-CHINA, NWU-CHINA-A and WS-HZBIOX that do gut probiotics like us to discuss safety issues and measures related to the project.

Intestinal Alliance

In the Gut Alliance, the NWU-CHINA-A team and we have publicized the impact of Helicobacter pylori on our human body and preventive measures. It has a wide transmission range, strong adsorption capacity, and is not easy to detect after infection. It is transmitted in a way that is oral to mouth and from mouth to feces. People can go to the regular hospital for examination, the current commonly used detection methods are breath test and gastroscopy, the simplest and most convenient test method is C14 or C13 breath test, you only need to exhale gas against a bag to seal the bag, the rest will be handed over to the medical staff, patiently wait. Although Helicobacter pylori can be eradicated, it is extremely contagious and is prevented by:

1) Wash your hands before and after going to the toilet: hand washing should focus on cleaning the palms of your hands, the back of your hands and the gaps between your fingertips, and don't give Helicobacter pylori a chance.

2) Food must go through high temperatures: Helicobacter pylori has a weakness, that is, it is not heat-resistant, water must be boiled to drink, meat must be cooked to eat, milk should be sterilized to drink.

3) Eat less irritating foods: eat less and more meals, do not smoke, do not drink, eat a balanced diet, chew slowly and swallow.

4) It is recommended to divide meals: patients with infection at home should choose to use public chopsticks until they are completely cured.

5) Mouth-to-mouth feeding is prohibited

6) Replace the dental appliance regularly: It is recommended to use mouthwash and bacteriostatic toothpaste for a period of time to relieve oral inflammation, and the toothbrush is changed once every three months.

Helicobacter pylori

We also work with Worldshaper_HZBIOX, and the knowledge they share about the relationship between gut microbiota and sugar substitutes enriches our safety knowledge and that of the general public. Sugar substitutes are also known as aspartame (containing phenylalanine). It is a sugar substitute commonly used in low-sugar beverages. It is considered a neurotoxin that damages the nervous system, so these symptoms are mainly related to the nervous system. Some of the common symptoms associated with SPLENDa, including headaches, memory loss, epilepsy, vision loss, coma, and cancer, can disrupt the balance of the intestinal flora, induce lesions in the intestinal flora, increase pathogenic bacteria, and cause enteritis, diarrhea, psychiatric disorders, and cancer. Protecting the gut flora protects the brain's memory, cognition and immunity. Sugar substitutes will increase appetite and be more harmful to weight loss.

Aspartame

Reference

[1] Molin, S., Klemm, P., Poulsen, L. et al. Conditional Suicide System for Containment of Bacteria and Plasmids. Nat Biotechnol 5, 1315–1318 (1987). https://doi.org/10.1038/nbt1287-1315

[2] Jayashree, Sathyanarayanan, et al. “Genome-Wide Identification of Probiotic Escherichia Coli Nissle 1917 (ECN) Fitness Genes during Adhesion to the Intestinal Epithelial Cells Caco-2.” Gene, vol. 803, 2021, p. 145890., https://doi.org/10.1016/j.gene.2021.145890.

[3] Ruiz-Echevarría, M J et al. “The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co-ordinated action of the Kis and Kid proteins.” Molecular microbiology vol. 5,11 (1991): 2685-93. doi:10.1111/j.1365-2958.1991.tb01977.x

[4] Bravo, A et al. “Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1.” Molecular & general genetics : MGG vol. 215,1 (1988): 146-51. doi:10.1007/BF00331316

[5] Kamphuis, Monique B et al. “Structure and function of bacterial kid-kis and related toxin-antitoxin systems.” Protein and peptide letters vol. 14,2 (2007)

[6] 益生菌包埋术，让活性益生菌包被后活的更有价值新营养

[7]【农业转基因生物安全评价管理办法（2022年1月21日修订版）】http://www.moa.gov.cn/ztzl/zjyqwgz/zcfg/202206/t20220607_6401864.htm

[8] “How GMOs Are Regulated in the United States” food-agricultural-biotechnology U.S. Food ＆Drug Administration.

[9] France's National Assembly approves FATCA reciprocity amendment, citing 'accidentels' July 27, 2022By Helen Burggraf News

[10] Japan Updates Labeling Rules for Genetically Modified Foods FOOD SAFETY magazine July 8，2022

[11] Safety of genetically modified foods Safety assessment of GM foods in Canada Government of Canada. 2022-08-05

[12]https://www.allaboutfeed.net/animal-feed/raw-materials/french-gmo-labelling-law-to-hit-feed-producers/

[13] Everything you need to know about Canada's new laws for GMOs and gene editing By Marc Fawcett-Atkinson | News, Food Insider | March 11th 2021